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Cortical Num1p interacts with the dynein intermediate chain Pac11p and cytoplasmic microtubules in budding yeast.

Farkasovsky M, Küntzel H - J. Cell Biol. (2001)

Bottom Line: The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p.Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells.In addition, Num1p may transiently cooperate with an associated Bni1p-Kar9p complex at the bud tip of early anaphase cells.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck Institute for Experimental Medicine, D-37075 Göttingen, Germany.

ABSTRACT
Num1p, a cortical 313-kD protein, controls cytoplasmic microtubule (cMT) functions and nuclear migration through the bud neck in anaphase cells. A green fluorescent protein (GFP)-Num1p fusion protein localizes at the bud tip and the distal mother pole of living cells, apparently forming cMT capture sites at late anaphase. In addition, galactose-induced GFP-Num1p is seen at the bud neck and in lateral regions of the mother cortex. The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p. Num1p associates with the dynein intermediate chain Pac11p in the presence of Dyn1p, and with the alpha-tubulin Tub3p, as shown by coimmune precipitation of tagged proteins. Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells. Our data suggest that Num1p controls nuclear migration during late anaphase by forming dynein-interacting cortical cMT capture sites at both cellular poles. In addition, Num1p may transiently cooperate with an associated Bni1p-Kar9p complex at the bud tip of early anaphase cells.

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Coimmune precipitation of Myc-Num1p with HA-Pac11p (A) and HA-Tub3p (B). (A) Anti-Myc immunoprecipitates obtained from strains FMY684 (lanes 1 and 5), FMY899 (lanes 2 and 6), FMY905 (lanes 3 and 7), and FMY912 (lanes 4 and 8). (B) Anti-Myc immunoprecipitates obtained from strains FMY684 (lanes 1 and 4), FMY682 (lanes 2 and 5) and FMY603 (lanes 3 and 6) were probed with anti-HA (lanes 1–3) and anti-Myc (lanes 4–6) antibodies. Anti-HA immunoprecipitates obtained from strains FMY682 (lanes 7 and 10), FMY684 (lanes 8 and 11), and FMY603 (lanes 9 and 12) were probed with anti-Myc (lanes 7–9) and anti-HA (lanes 10–12) antibodies.
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Figure 7: Coimmune precipitation of Myc-Num1p with HA-Pac11p (A) and HA-Tub3p (B). (A) Anti-Myc immunoprecipitates obtained from strains FMY684 (lanes 1 and 5), FMY899 (lanes 2 and 6), FMY905 (lanes 3 and 7), and FMY912 (lanes 4 and 8). (B) Anti-Myc immunoprecipitates obtained from strains FMY684 (lanes 1 and 4), FMY682 (lanes 2 and 5) and FMY603 (lanes 3 and 6) were probed with anti-HA (lanes 1–3) and anti-Myc (lanes 4–6) antibodies. Anti-HA immunoprecipitates obtained from strains FMY682 (lanes 7 and 10), FMY684 (lanes 8 and 11), and FMY603 (lanes 9 and 12) were probed with anti-Myc (lanes 7–9) and anti-HA (lanes 10–12) antibodies.

Mentions: Fig. 7 A demonstrates that HA-Pac11p coprecipitates with Myc-Num1p in a wild-type background (lane 3, strain FMY905) but not in the absence of Dyn1p (lane 4, strain FMY912). Furthermore, the 60-kD HA-Pac11p band is absent from anti-Myc precipitates of cells expressing only HA-Pac11p (lane 2, strain FMY899), supporting the specificity of the coprecipitation. Lanes 5 to 8 of Fig. 7 A confirm the presence or absence of Myc-Num1p in the anti-Myc precipitates. Similarly, the results shown in Fig. 7 B indicate that HA-Tub3p (50 kD) coprecipitates with Myc-Num1p (Fig. 7, lane 3), and Myc-Num1p coprecipitates with HA-Tub3p (Fig. 7, lane 9). The specificity of these coprecipitations is documented in Fig. 7, lanes 2 and 8.


Cortical Num1p interacts with the dynein intermediate chain Pac11p and cytoplasmic microtubules in budding yeast.

Farkasovsky M, Küntzel H - J. Cell Biol. (2001)

Coimmune precipitation of Myc-Num1p with HA-Pac11p (A) and HA-Tub3p (B). (A) Anti-Myc immunoprecipitates obtained from strains FMY684 (lanes 1 and 5), FMY899 (lanes 2 and 6), FMY905 (lanes 3 and 7), and FMY912 (lanes 4 and 8). (B) Anti-Myc immunoprecipitates obtained from strains FMY684 (lanes 1 and 4), FMY682 (lanes 2 and 5) and FMY603 (lanes 3 and 6) were probed with anti-HA (lanes 1–3) and anti-Myc (lanes 4–6) antibodies. Anti-HA immunoprecipitates obtained from strains FMY682 (lanes 7 and 10), FMY684 (lanes 8 and 11), and FMY603 (lanes 9 and 12) were probed with anti-Myc (lanes 7–9) and anti-HA (lanes 10–12) antibodies.
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Figure 7: Coimmune precipitation of Myc-Num1p with HA-Pac11p (A) and HA-Tub3p (B). (A) Anti-Myc immunoprecipitates obtained from strains FMY684 (lanes 1 and 5), FMY899 (lanes 2 and 6), FMY905 (lanes 3 and 7), and FMY912 (lanes 4 and 8). (B) Anti-Myc immunoprecipitates obtained from strains FMY684 (lanes 1 and 4), FMY682 (lanes 2 and 5) and FMY603 (lanes 3 and 6) were probed with anti-HA (lanes 1–3) and anti-Myc (lanes 4–6) antibodies. Anti-HA immunoprecipitates obtained from strains FMY682 (lanes 7 and 10), FMY684 (lanes 8 and 11), and FMY603 (lanes 9 and 12) were probed with anti-Myc (lanes 7–9) and anti-HA (lanes 10–12) antibodies.
Mentions: Fig. 7 A demonstrates that HA-Pac11p coprecipitates with Myc-Num1p in a wild-type background (lane 3, strain FMY905) but not in the absence of Dyn1p (lane 4, strain FMY912). Furthermore, the 60-kD HA-Pac11p band is absent from anti-Myc precipitates of cells expressing only HA-Pac11p (lane 2, strain FMY899), supporting the specificity of the coprecipitation. Lanes 5 to 8 of Fig. 7 A confirm the presence or absence of Myc-Num1p in the anti-Myc precipitates. Similarly, the results shown in Fig. 7 B indicate that HA-Tub3p (50 kD) coprecipitates with Myc-Num1p (Fig. 7, lane 3), and Myc-Num1p coprecipitates with HA-Tub3p (Fig. 7, lane 9). The specificity of these coprecipitations is documented in Fig. 7, lanes 2 and 8.

Bottom Line: The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p.Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells.In addition, Num1p may transiently cooperate with an associated Bni1p-Kar9p complex at the bud tip of early anaphase cells.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck Institute for Experimental Medicine, D-37075 Göttingen, Germany.

ABSTRACT
Num1p, a cortical 313-kD protein, controls cytoplasmic microtubule (cMT) functions and nuclear migration through the bud neck in anaphase cells. A green fluorescent protein (GFP)-Num1p fusion protein localizes at the bud tip and the distal mother pole of living cells, apparently forming cMT capture sites at late anaphase. In addition, galactose-induced GFP-Num1p is seen at the bud neck and in lateral regions of the mother cortex. The bud tip location of Num1p depends on Bni1p but does not require Kar9p, Dyn1p, or cMTs, whereas cMT contacts with polar Num1p dots are reduced in cells lacking Dyn1p. Num1p associates with the dynein intermediate chain Pac11p in the presence of Dyn1p, and with the alpha-tubulin Tub3p, as shown by coimmune precipitation of tagged proteins. Num1p also forms a complex with Bni1p and Kar9p, although Num1p is not required for Bni1p- and Kar9p-dependent nuclear migration to the bud neck in preanaphase cells. Our data suggest that Num1p controls nuclear migration during late anaphase by forming dynein-interacting cortical cMT capture sites at both cellular poles. In addition, Num1p may transiently cooperate with an associated Bni1p-Kar9p complex at the bud tip of early anaphase cells.

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